Abstract Deciphering the mechanisms controlling cell fate choice and maintenance in the brain is a critical step in understanding devastating neurological disorders such as microcephaly, schizophrenia and brain cancer that result from defects in these processes. An unresolved question is how the mammalian neuronal identity is maintained and protected, particularly at the transcriptional level, during development and adult life. The BTB/POZ and Zinc finger transcription factor RP58 (aka ZBTB18), is required for brain development and neuronal differentiation both in vivo and in vitro; Mutations in RP58 are linked to human microcephaly and corpus callosum agenesis. We have analyzed the embryonic cortical post mitotic neurons? transcriptome and showed that the expression of gene markers of other cell lineages, such as myogenic lineage, is increased following Rp58 deletion suggesting that RP58 protects the neuronal identity by repressing genes of other lineages. Our overall hypothesis is that RP58 is required to establish and maintain the neuronal identity and that the loss of its transcriptional function may lead to microcephaly and to neurodegenerative diseases in the adult brain. To decipher how RP58 controls cell differentiation in the brain and to identify RP58 protein partners that may be involved in its function, we have performed RP58 immunoprecipitation coupled to mass spectrometry (IP-MS) experiments on mouse embryonic and postnatal cortices. Our data show that RP58 binds to members of both the Polycomb Repressive Complex 2 (PRC2) and the SWI/SNF complex, two critical chromatin remodeling complexes involved in brain development. In addition, using quantitative histone proteomics and western blot analyses, we show that deletion of Rp58 in the embryonic brain leads to global changes in histones post- translational modifications (PTMs) including to decreased H3K27 methylation (H3K27me) and increased H3K9/14 acetylation. These results raise the hypothesis that RP58 controls the identity of developing and adult neurons and thus their transcriptional program by modulating the PRC2 and SWI/SNF complexes function and the chromatin landscape. The following aims will address these hypotheses. Aim 1: Decipher how RP58 and chromatin remodeling complexes interact to establish and maintain neuronal identity. Aim 2: Decipher if and how RP58 is required for histone PTMs in neural cells. Aim 3: Determine the cell and developmental stage-specific RP58 protein-protein interacting network in neural cells. This grant is a collaboration between the Dahmane lab (Weill Cornell Medicine) with expertise in developmental neurobiology and mouse genetics and the Garcia lab (University of Pennsylvania) with expertise in quantitative mass spectrometry as it relates to epigenetic mechanisms and chromatin regulation. Completion of these proposed studies will lead to the elucidation of the cell type and stage specific RP58 regulatory mechanisms controlling neuronal identity and to key insights into the genetic and epigenetic regulation of brain growth and of their misregulation in brain disorders such as microcephaly.